Saposin C-DOPS: A Novel Anti-Tumor Agent

ABSTRACT

Compositions and methods for treating subjects with disorders characterized by hyper-proliferating cells such as tumors and cancers are provided. The compositions comprise agents that are combinations of saposin C (or prosaposin-related polypeptides) and phospholips (or inner leaflet components). This anti-tumor agent is administered in the methods of the invention according to a dosing regimen. Administering an agent of the invention results in a positive therapeutic response in a subject with a tumor.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority as a divisional application to U.S.patent application Ser. No. 12/332,809, filed on Dec. 11, 2008, which isa continuation of U.S. patent application Ser. No. 10/801,517, filed onMar. 16, 2004, which claims priority to and benefit of U.S. ProvisionalApplication No. 60/466,166, filed on Apr. 28, 2003, which applicationsare hereby incorporated by reference in their entirety for all purposes.

GOVERNMENT GRANT INFORMATION

This invention was made with government support under DK057690 awardedby the National Institutes of Health. The government has certain rightsin the invention.

FIELD OF THE INVENTION

The present invention is directed to compositions for and methods ofmodulating proliferating cell volume, more particularly to modulatingtumor and cancer volume. Additionally the invention is directed tocompositions for and methods of treating cancer.

BACKGROUND OF THE INVENTION

Saposins, a family of small (˜80 amino acids), heat stableglycoproteins, are essential for the in vivo hydrolytic activity ofseveral lysosomal enzymes in the catabolic pathway of glycosphingolipids(see Grabowski et al. (1990) Crit. Rev. Biochem. Mol. Biol. 25:385-414;Furst et al. (1992) Biochim. Biophys. Acta. 1126:1-16; Kishimoto et al.(1992) J Lipid Res. 33:1255-1267). Four members of the saposin family(A, B, C, and D) are proteolytically hydrolyzed from a single precursorprotein, prosaposin (see Fujibayashi et al. (1985) Am. J Hum. Genet.37:741-748; O'Brien et al. (1988) Science 241:1098-1101; Rorman et al.(1989) Genomics 5:486-492; Nakano et al. (1989). J Biochem. (Tokyo) 105:152-154; Reiner et al. (1989) J Mol. Neurosci. 1:225-233; hereinincorporated by reference. The complete amino acid sequences forsaposins A, B, C, and D have been reported as well as the genomicorganization and cDNA sequence of prosaposin (see Fujibayashi et ai.(1985) Am. J Hum. Genet. 37:741-748; O'Brien et ai. (1988) Science241:1098-1101; Rorman et al. (1989) Genomics 5:486-492).

Saposins are defined as sphingolipid activator proteins or coenzymes.Structurally, saposins A, B, C, and D have approximately 50-60%similarity including six strictly conserved cysteine residues (see Furstet al. (1992) Biochim. Biophys. Acta 1126:1-16) that form threeintradomain disulfide bridges whose placements are identical (seeVaccaro et al. (1995) J. Biol. Chem. 270:9953-9960). All saposinscontain one glycosylation site with conserved placement in theN-terminal sequence half, but glycosylation is not essential to theiractivities (see Qi et al. (1998) Biochemistry 37:11544-11554 and Vaccaroet al. (1995) J. Biol. Chem. 270:30576-30580, herein incorporated byreference in their entirety).

All saposins and saposin-like proteins and domains contain a “saposinfold” when in solution. This fold is a multiple α-helical bundle motif,characterized by three conserved disulfide structures and severalamphipathic polypeptides. Despite this shared saposin-fold in solution,saposins and saposin-like proteins have diverse in vivo biologicalfunctions in the enhancement of lysosomal sphingolipid (SL) andglycosphingolipid (GSL) degradation by specific hydrolases. Because ofthese roles, the saposins occupy a central position in the control oflysosomal sphingolipid and glycosphingolipid metabolisms (see Kishimotoet al. (1992) J. Lipid Res. 33:1255-1267; Fujibayashi et al. (1985) Am.J. Hum. Genet. 37:741-748; O'Brien et al. (1988) Science 241:1098-1101,herein incorporated by reference). In addition, saposins participate inthe fusion and destabilization of acidic phospholipid vesicles (seeVaccaro et al. (1994) FEBS Letters 349:181-186, herein incorporated byreference).

Saposin C is required for optimal hydrolysis of glucosylceramide by acidβ-glucosidase (Gcase, EC 3.1.2.45) in vivo and in vitro. Also, saposin Cinduced fusion toward phosphatidylserine containing vesicles has beenobserved by electron microscopy (see Vaccaro et al. (1994) FEBS Letters349:181-186, herein incorporated by reference). Further, saposin C hasthe general property of lipid membrane binding activity or plasmamembrane affinity. Saposins associate with lipid membranes by embeddinginto the outer leaflets. The H-1 and H-5 helices are integral to thisprocess, suggesting that proper membrane interaction of saposin Caffects its specificity and activity. In addition, saposin C inducesstructural changes of the membrane. The dynamic processes of saposininteractions with planar phospholipid bilayers have been visualized inreal time using atomic force microscopy (see Qi et al. (2001) J. Biol.Chem. 276:27010-27017 and You et al. (2001) FEBS Lett. 503:97-102,herein incorporated by reference).

Phospholipid asymmetry is a well-known characteristic of mammalianplasma membranes. The outer leaflet of the lipid bilayer is rich incholine-phospholipids, whereas aminophospholipids are preferentially inthe inner leaflet (Bevers et al. (1998) Lupus Suppl. 2:S126-S131).Phosphatidylserine (PS) and phosphatidylethanolamine (PE) reside almostexclusively in the inner leaflet, and phosphatidylcholine (PC) andsphingomyelin are enriched in the outer leaflet. Phospholipid asymmetrymight be a general property of all cells (Woon et al. (1999) CellCalcium 25(4):313-320). The plasma membrane phospholipid asymmetry ismaintained through a variety of mechanisms, including aminophospholipidtranslocases and phospholipid scramblases (U.S. Patent Application No:20020081698).

In general, tumor or cancer cells grow rapidly in comparison to normalcells. These abnormal cells produce a significant amount of protonsprimarily by generating lactic acid during glycolysis or by generatingcarbon dioxide during respiration due to a fast metabolic rate.Therefore, the surrounding sites of these cells and tissues are usuallyfound to be more acidic than those of cells with a normal growth rate.

Squamous cell carcinomas (SCCs) of the skin are one of the most commonskin cancers associated with a substantial risk of metastasis (Alam etal. (2001) N. Engl. J. Med. 344:975-983, herein incorporated byreference). Cancers of the skin are classified into two categories,melanoma and non-melanoma skin cancers (NMSC). According to theestimation by the American Cancer Society, more than one million casesof NMSC are found in the United States each year. SCC accounts forapproximately 20% of all cutaneous tumors and there are about 200,000new SCC cases in the United States each year. SCC is the most frequentform of malignant tumor in the transition from the skin to the mucosaand in the mucosa itself (Boni et al. (2002) Neuroendocrinology Letters23S2:48-51). The current treatments of SCC patients includeelectrodessication and curettage, excision, cryotherapy, surgicalexcision, or Mohs' surgery. Appropriate use of electrodessication andcurettage, excision, or cryotherapy can eliminate small (<1 cm indiameter), well-defined tumors with a low risk of metastasis. Surgicalexcision and Mohs' surgery offer the highest rates of cure for patientswith high-risk primary or recurrent SCCs. However, these treatments aremore costly with the risk of hematoma, seroma, infection, and wounddehiscence.

Thus, development of an effective, low-cost SCC treatment with improvedcosmetic outcomes is desirable. It is also of importance to develop aneffective, low-cost treatment for other cancer types such as breast andprostate cancers and lymphomas.

SUMMARY OF THE INVENTION

Compositions and methods for modulating distribution of components ofthe inner and outer leaflets of plasma membranes are provided. Agents ofthe invention comprise an inner leaflet component and a prosaposinrelated polypeptide. By “inner leaflet component” is intended anymolecule or structural analog thereof naturally occurring in the innerleaflet of a plasma membrane of a cell, particularly an animal cell,more particularly a mammalian cell. In an embodiment the inner leafletcomponent is phosphatidylserine or a structural analog thereof, such asdioleoylphosphatidylserine (DOPS). The amino acid sequence of prosaposinis set forth in SEQ ID NO:1 in the sequence listing. Prosaposin relatedpolypeptides share at least 80% identity to the amino acid sequence setforth in SEQ ID NO:1 or a fragment thereof and retain plasma-membraneaffinity. In an embodiment, the prosaposin related polypeptide issaposin C (SEQ ID NO:2 of the sequence listing) or a saposin C-relatedpolypeptide. Saposin C-related polypeptides share at least 80% identityto the amino acid sequence set forth in SEQ ID NO:2 and retainplasma-membrane affinity. The molar ratio of the polypeptide to theinner leaflet component in an agent of the invention is in the rangefrom about 1:1 to about 1:50, preferably about 1:1 to about 1:25, morepreferably about 1:1 to about 1:10, yet more preferably about 1:7 orabout 1:3. In an embodiment, agents of the invention further comprise apharmaceutically acceptable carrier. An agent of the invention promotescell death, such as cell death through apoptosis. In an embodiment, theagent preferentially induces in apoptosis in hyper-proliferating cellssuch as, but not limited to, tumor and cancer cells. Thus, in anembodiment of the invention, the agent is an anti-tumor agent. In anaspect of the invention, the agent preferentially induces apoptosis incancer cells such as, but not limited to, sarcoma cells, neuroblastomacells, and squamous cell carcinoma cells.

Methods for modulating the distribution of an inner leaflet component inthe plasma membrane of a cell of a subject are provided. Such methodsinvolve administering an agent comprising an inner leaflet component anda prosaposin related polypeptide to a subject. The methods alter thedistribution of the inner leaflet component in the outer leaflet of aplasma membrane of a cell of the subject. In an embodiment, theconcentration of the inner leaflet component in the outer leaflet isincreased. In an embodiment of the invention, modulating thedistribution of an inner leaflet component preferentially occurs inhyper-proliferating cells of the subject. Preferably suchhyper-proliferating cells are tumor cells or cancer cells. In an aspectof the invention, modulating the concentration of an inner leafletcomponent in the outer leaflet of the plasma membrane induces apoptosis.

Methods for modulating tumor volume in a subject are provided. Suchmethods involve administering an agent comprising an inner leafletcomponent and a prosaposin related polypeptide to a subject. The agentmay further comprise a pharmaceutically acceptable carrier. Suitablesubjects include mammals, particularly humans, with tumors. In anembodiment, the agent promotes cell death in hyper-proliferating cells.Cell death may occur through apoptosis. Any tumor is a potential targetof the invention. The target tumors contain hyper-proliferating cellssuch as tumor cells and cancer cells. Such target tumors include, butare not limited to, sarcomas, neuroblastomas, and squamous cellcarcinomas. In an embodiment, modulating the tumor volume results in adecrease in tumor volume. It is envisioned that the death ofhyper-proliferating cells within the tumor results in a decrease intumor volume.

Methods for treating a cancer in a subject are provided. Such methodsinvolve administering an agent comprising an inner leaflet component anda prosaposin related polypeptide to a subject. The agent may furthercomprise a pharmaceutically acceptable carrier. Suitable subjectsinclude mammals, particularly humans, with cancers. In an embodiment,the agent promotes cell death in hyper-proliferating cells. Cell deathoccurs through a process such as, but not limited to, apoptosis. Anycancer is a potential target of the invention. Target cancers containhyper-proliferating cells such as tumor and cancer cells. Target cancercells include, but are not limited to, cells derived from sarcomas,neuroblastomas, breast carcinomas, and squamous cell carcinomas. In anaspect of the invention, the agent is administered enterally,parenterally, subcutaneously, intravenously, intraperitoneally, ortopically. Either a single or multiple doses of the agent may beadministered to a subject to treat a cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents normal immortalized keratinocytes (NIK) (Panels A and B)and squamous cell carcinoma cells (SCC) (Panels C and D). The cells inPanels A and C received placebo treatments. The cells in Panels B and Dwere treated with an agent of the invention comprising 8 μM saposin Cand 26 μM DOPS. Details of the experiment are described elsewhereherein.

FIG. 2 presents murine lymphoma cells from the L5178Y-R cell line. Thecells in Panel A received a placebo treatment. The cells in Panel B weretreated with 60 μM DOPS. The cells in Panel C were treated with 20 μMsaposin C. The cells in Panel D were treated with an agent of theinvention comprising 10 μM saposin C and 30 μM DOPS.

FIG. 3 presents results obtained from assessment of the average tumorvolume on nude mice bearing human squamous cell carcinoma xenograftsprior to and subsequent to subcutaneous injection of either a placebo(phosphate buffered saline, indicated with solid triangles and dashedlines) or agent of the invention (saposin C at 10 mg/Kg body weight/DOPSat 2 mg/Kg body weight, indicated with solid squares and lines). Errorbars indicate the standard error. Tumor volume is indicated in mm³, andtime is indicated as days of tumor growth. Panel A presents resultsobtained from mice treated twice. The days of the first and secondinjections are indicated with arrows. Panel B presents results obtainedfrom mice treated once. The injection day is indicated with an arrow.Details of the experiments are described elsewhere herein.

FIG. 4 presents fluorescent micrographs of human squamous cell carcinomatumor tissues from xenografts. The cells in Panel A were treated with afluorescently labeled mixture of phosphatidylserine and DOPS(NBD-DOSP/DOPS). The cells in Panel B were treated with a fluorescentlylabeled mixture of phosphatidylserine, DOPS, and saposin C. Details ofthe experiment are described elsewhere herein.

FIG. 5 presents micrographs of human squamous cell carcinoma tumortissues from xenografts. Tissues were obtained from tumors treated witheither DOPS (2 mg/kg of body weight, Panels A and C) or saposin C (10mg/kg of body weight) and DOPS (2 mg/kg of body weight, Panels B and D).The tissues were stained with TUNEL staining (Panels A and B) orhematoxylin and eosin (Panels C and D). The arrows in Panel B indicateapoptotic cells. The dark staining in Panel D indicates area ofnecrosis.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to compositions and methods for modulatingthe distribution of inner leaflet components in a plasma membrane.Further, the present invention relates to compositions and methods formodulating hyper-proliferating cells, particularly disorders involvinghyper-proliferating cells, more particularly tumors and cancers such assquamous cell carcinomas and lymphomas. The compositions comprise anagent with a prosaposin-related polypeptide, particularly saposin C, andan inner leaflet component, particularly a phosphatidylserine orstructural analog thereof, more particularly dioleoylphosphatidylserine(DOPS). Combinations of these two compounds exhibit anti-tumor activityand hence are referred to as anti-tumor agents. By “anti-tumor activity”is intended a reduction in the rate of cell proliferation, and hence adecline in the growth rate of an existing tumor or in a tumor thatarises during therapy, and/or destruction of existing neoplastic (tumor)cells or newly formed neoplastic cells, and hence a decrease in theoverall size of a tumor during therapy. Treatment with a combination ofsaposin C (or a prosaposin-related polypeptide) and DOPS (or an innerleaflet component) causes a physiological response that modulates thedistribution of an inner leaflet component in the plasma membrane.

The anti-tumor activity of an agent of the invention is not limited to aparticular mode of action, but may function through a variety of modesof action including but not limited to, apoptosis. Environmental factorscontribute to the agent's preferential effect on tumor cells. Theseenvironmental factors include, but are not limited to, lower pH levelsnear tumor cells, increasing hypoxic conditions, and altered lipidpresentation on the outer membrane of tumor cells. Hypoxia is anepigenetic factor that stimulates expression and release of vascularendothelial growth factor (VEGF) from tumor cells. VEGF is known in theart as a vascular permeability factor and may play a role in thedisorganized and leaky vasculatures of tumor tissues as compared tonormal vasculature. In an embodiment, a saposin C-DOPS agent of theinvention preferentially penetrates tumor tissue rather than healthytissue after intravenous administration.

The invention encompasses isolated or substantially purified protein orpolypeptide compositions. An “isolated” or “purified” polypeptide orbiologically active portion thereof, is substantially or essentiallyfree from components that normally accompany or interact with theprotein as found in its naturally occurring environment. Thus, anisolated or purified protein is substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized. A protein that is substantially free of cellularmaterial includes preparations of protein having less than about 30%,20%, 10%, 5%, or 1% (by dry weight) of contaminating protein. When theprotein of the invention or biologically active portion thereof isrecombinantly produced, preferably culture medium represents less thanabout 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors ornon-protein-of-interest chemicals.

As used herein, a “prosaposin-related polypeptide” is any polypeptidehaving the prosaposin amino acid sequence set forth in SEQ ID NO:1, anamino acid sequence at least 80% identical to the amino acid sequenceset forth in SEQ ID NO:1, or a proteolytically processed fragmentthereof, wherein said polypeptide retains plasma membrane affinity.Fragments and variants of the prosaposin polypeptide (SEQ ID NO: 1) arealso encompassed by the present invention. Prosaposin is proteolyticallyprocessed into four saposins, saposins A, B, C, and D. As used herein, a“saposin C-related polypeptide” is any polypeptide having the saposin Camino acid sequence set forth in SEQ ID NO:2 or an amino acid sequenceat least 80% identical to the amino acid sequence set forth in SEQ IDNO:2, wherein said polypeptide retains plasma membrane affinity.Fragments and variants of the saposin C-related polypeptide (SEQ IDNO:2) are also encompassed by the present invention. By “fragment” isintended a portion of the amino acid sequence and hence protein.Prosaposin protein fragments retain the biological activity ofprosaposin and hence possess plasma membrane affinity. Saposin C proteinfragments retain the biological activity of saposin C and hence possessplasma membrane affinity. As used herein “plasma membrane affinity”refers to an ability to interact with phospholipid surfaces throughelectrostatic or hydrophobic interactions.

A fragment of a biologically active portion of prosaposin polypeptide ofthe invention will encode at least 15, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330,340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470,480, 490, 500, 510, 520 continuous amino acids, or up to 524 amino acidspresent in a prosaposin polypeptide of the invention. A fragment of abiologically active portion of saposin C polypeptide of the inventionwill encode at least 15, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or80 contiguous amino acids present in a saposin C polypeptide of theinvention.

By “variants” is intended substantially similar sequences. Fornucleotide sequences, conservative variants include those sequencesthat, because of the degeneracy of the genetic code, encode the aminoacid sequence of a prosaposin polypeptide of the invention. Naturallyoccurring allelic variants such as these can be identified with the useof well-known molecular biology techniques, as, for example, withpolymerase chain reaction (PCR) and hybridization techniques. Variantnucleotide sequences also include synthetically derived nucleotidesequences, such as those generated, for example, by using site-directedmutagenesis but which still encode a prosaposin.

By “variant” protein is intended a protein derived from the nativeprotein by deletion (so-called truncation) or addition of one or moreamino acids to the N-terminal and/or C-terminal end of the nativeprotein; deletion or addition of one or more amino acids at one or moresites in the native protein; substitution of one or more amino acids atone or more sites in the native protein, or synthetically producedpolypeptides having such an amino acid sequence. Variant proteinsencompassed by the present invention are biologically active, that isthey continue to possess the desired biological activity of the nativeprotein, that is, plasma membrane affinity as described herein. Suchvariants may result from, for example, genetic polymorphism or fromhuman manipulation. Biologically active variants of a native prosaposinprotein of the invention will have at least about 80%, 85%, preferablyat least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, and morepreferably at least about 98%, 99% or more sequence identity to theamino acid sequence for the native protein as determined by sequencealignment programs described elsewhere herein using default parameters.A biologically active variant of a protein of the invention may differfrom that protein by as few as 1-15 amino acid residues, as few as 1-10,such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acidresidue.

The proteins of the invention may be altered in various ways includingamino acid substitutions, deletions, truncations, and insertions.Methods for such manipulations are generally known in the art. Forexample, amino acid sequence variants of the prosaposin protein can beprepared by mutations in the DNA. Methods for mutagenesis and nucleotidesequence alterations are well known in the art. See, for example, Kunkel(1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987)Methods in Enzymol. 154:367-382; U.S. Pat. No. 4,873,192; Walker andGaastra, eds. (1983) Techniques in Molecular Biology (MacMillanPublishing Company, New York) and the references cited therein. Guidanceas to appropriate amino acid substitutions that do not affect biologicalactivity of the protein of interest may be found in the model of Dayhoffet al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed.Res. Found., Washington, D.C.) and Qi et al. (2001) J. Biol. Chem.276:27010-27017, herein incorporated by reference. Conservativesubstitutions, such as exchanging one amino acid with another havingsimilar properties, may be preferable.

Thus, the polypeptide and amino acid sequences of the invention includeboth the naturally occurring sequences as well as mutant forms,variants, and modified forms thereof Such variants will continue topossess the desired plasma membrane affinity activity. Obviously, themutations that will be made in the DNA encoding the variant must notplace the sequence out of reading frame and preferably will not createcomplementary regions that could produce secondary mRNA structure. See,EP Patent Application Publication No. 75,444.

The deletions, insertions, and substitutions of the protein sequenceencompassed herein are not expected to produce radical changes in thecharacteristics of the protein. However, when it is difficult to predictthe exact effect of the substitution, deletion, or insertion in advanceof doing so, one skilled in the art will appreciate that the effect willbe evaluated by routine screening assays. That is, the plasma membraneaffinity can be evaluated by methods known in the art including, but notlimited to, fluorescence spectrophotometry, fluorescence resonanceenergy transfer, or circular dichroism measurements. See, for example,Qi et al. (2001) J. Biol. Chem. 276:27010-27017, herein incorporated byreference.

Variant proteins also encompass proteins derived from a mutagenic andrecombinogenic procedure such as DNA shuffling. With such a procedure,one or more different saposin C sequences can be manipulated to create anew saposin C possessing the desired properties. In this manner,libraries of recombinant polynucleotides are generated from a populationof related sequence polynucleotides comprising sequence regions thathave substantial sequence identity and can be homologously recombined invitro or in vivo. For example, using this approach, sequence motifsencoding a domain of interest may be shuffled between the prosaposingene of the invention and other known prosaposin genes to obtain a newgene coding for a protein with an improved property of interest, such asan altered plasma membrane affinity. Strategies for such DNA shufflingare known in the art. See, for example, Stemmer (1994) Proc. Natl. Acad.Sci. USA 91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri etal. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol.272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and U.S. Pat.Nos. 5,605,793 and 5,837,458.

The following terms are used to describe the sequence relationshipsbetween two or more amino acid sequences or polypeptides: (a) “referencesequence”, (b) “comparison window”, (c) “sequence identity”, (d)“percentage of sequence identity”, and (e) “substantial identity”.

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length polypeptide or amino acid sequence or the completepolypeptide sequence.

(b) As used herein “comparison window” makes reference to a contiguousand specified segment of an amino acid sequence, wherein the amino acidsequence in the comparison window may comprise additions or deletions(i.e. gaps) compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences.Generally the comparison window is at least 20 contiguous amino acids inlength, and optionally can be 30, 40, 50, 100, or longer. Those of skillin the art understand that to avoid a high similarity to a referencesequence due to inclusion of gaps in the amino acid sequence a gappenalty is typically introduced and is subtracted from the number ofmatches.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent sequence identity between anytwo sequences can be accomplished using a mathematical algorithm.Preferred, non-limiting examples of such mathematical algorithms are thealgorithm of Myers and Miller (1988) CABIOS 4:11-17; the local homologyalgorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the homologyalignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol.48:443-453; the search-for-similarity-method of Pearson and Lipman(1988) Proc. Natl. Acad. Sci. 85:2444-2448; the algorithm of Karlin andAltschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, modified as inKarlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity. Forpurposes of the present invention, comparison of nucleotide or proteinsequences for determination of percent sequence identity to thesequences disclosed herein is preferably made using the GCG program GAP(Version 10.00 or later) with its default parameters or any equivalentprogram. By “equivalent program” is intended any sequence comparisonprogram that, for any two sequences in question, generates an alignmenthaving identical nucleotide or amino acid residue matches and anidentical percent sequence identity when compared to the correspondingalignment generated by the preferred program.

Sequence comparison programs include, but are not limited to: CLUSTAL inthe PC/Gene program (available from Intelligenetics, Mountain View,Calif.); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Version 8(available from Genetics Computer Group (GCG), 575 Science Drive,Madison, Wis., USA). Alignments using these programs can be performedusing the default parameters. The CLUSTAL program is well described byHiggins et al. (1988) Gene 73:237-244 (1988); Higgins et al. (1989)CABIOS 5:151-153; Corpet et al. (1988) Nucleic Acids Res. 16:10881-90;Huang et al. (1992) CABIOS 8:155-65; and Pearson et al. (1994) Meth.Mol. Biol. 24:307-331. The ALIGN program is based on the algorithm ofMyers and Miller (1988) supra. A PAM 120 weight residue table, a gaplength penalty of 12, and a gap penalty of 4 can be used with the ALIGNprogram when comparing amino acid sequences. The BLAST programs ofAltschul et al. (1990) J. Mol. Biol. 215:403 are based on the algorithmof Karlin and Altschul (1990) supra. BLAST nucleotide searches can beperformed with the BLASTN program, score=100, wordlength=12, to obtainnucleotide sequences homologous to a nucleotide sequence encoding aprotein of the invention. BLAST protein searches can be performed withthe BLASTX program, score=50, wordlength=3, to obtain amino acidsequences homologous to a protein or polypeptide of the invention. Toobtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST2.0) can be utilized as described in Altschul et al. (1997) NucleicAcids Res. 25:3389. Alternatively, PSI-BLAST (in BLAST 2.0) can be usedto perform an iterated search that detects distant relationships betweenmolecules. See Altschul et al. (1997) supra. When utilizing BLAST,Gapped BLAST, PSI-BLAST, the default parameters of the respectiveprograms (e.g., BLASTN for nucleotide sequences, BLASTX for proteins)can be used. National Center for Biotechnology Information (NCBI).Alignment may also be performed manually by inspection.

(c) As used herein, “sequence identity” or “identity” in the context oftwo nucleic acid or polypeptide sequences makes reference to theresidues in the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity”. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

(e)(i) The term “substantial identity” of polynucleotide sequences meansthat a polynucleotide comprises a sequence that has at least 70%sequence identity, preferably at least 80%, more preferably at least90%, and most preferably at least 95%, compared to a reference sequenceusing one of the alignment programs described using standard parameters.One of skill in the art will recognize that these values can beappropriately adjusted to determine corresponding identity of proteinsencoded by two nucleotide sequences by taking into account codondegeneracy, amino acid similarity, reading frame positioning, and thelike. Substantial identity of amino acid sequences for these purposesnormally means sequence identity of at least 60%, more preferably atleast 70%, 80%, 90%, and most preferably at least 95%.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other under stringent conditions.Generally, stringent conditions are selected to be about 5° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength and pH. However, stringent conditions encompasstemperatures in the range of about 1° C. to about 20° C. lower than theT_(m), depending upon the desired degree of stringency as otherwisequalified herein. Nucleic acids that do not hybridize to each otherunder stringent conditions are still substantially identical if thepolypeptides they encode are substantially identical. This may occur,e.g., when a copy of a nucleic acid is created using the maximum codondegeneracy permitted by the genetic code. One indication that twonucleic acid sequences are substantially identical is when thepolypeptide encoded by the first nucleic acid is immunologically crossreactive with the polypeptide encoded by the second nucleic acid.

(e)(ii) The term “substantial identity” in the context of a peptideindicates that a peptide comprises a sequence with at least 70% sequenceidentity to a reference sequence, preferably 80%, more preferably 85%,most preferably at least 90% or 95% sequence identity to the referencesequence over a specified comparison window. Preferably, optimalalignment is conducted using the homology alignment algorithm ofNeedleman and Wunsch (1970) J. Mol. Biol. 48:443-453. An indication thattwo peptide sequences are substantially identical is that one peptide isimmunologically reactive with antibodies raised against the secondpeptide. Thus, a peptide is substantially identical to a second peptide,for example, where the two peptides differ only by a conservativesubstitution. Peptides that are “substantially similar” share sequencesas noted above except that residue positions that are not identical maydiffer by conservative amino acid changes.

Agents of the invention comprise a prosaposin-like polypeptide and aninner leaflet component. By “inner leaflet component” is intended anymolecule or structural analog thereof naturally occurring in the innerleaflet of a plasma membrane of a cell, particularly an animal cell,more particularly a mammalian cell. In general the concentration of aninner leaflet component in the inner leaflet will be greater than theconcentration of that inner leaflet component in the outer leaflet. Itis recognized that during certain cellular perturbations such asapoptosis, necrosis, and hyperproliferative growth the normalcomposition of the inner and outer leaflets are altered. Exemplary innerleaflet components include, but are not limited to, phosphatidylserine,phosphatidylethanolamine, and structural analogs thereof. By “structuralanalog” of phosphatidylserine is intended any anionic phospholipid oracid lipid with a negatively charged head group including, but notlimited to, phosphatidic acid, phosphatidylglycerol,phosphatidylinositol, palmitoyloleoylphosphatidylserine,palmitelaidoyloleoylphosphatidylserine,myristoleoyloleoylphosphatidylserine, dilinoleoylphosphatidylserine,palmiticlinoleoylphosphatidylserine, lysophosphatidylserine, anddioleoylphosphatidylserine.

In an embodiment the compositions and methods of the invention aredirected toward modulating inner leaflet component distribution in aplasma membrane. In another embodiment, the compositions and methods ofthe invention are directed to the modulation and treatment of disordersinvolving hyper-proliferating cells such as tumors and cancers. By“modulate” is intended alter, change, vary, modify, or permute by atleast 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.Modulating the distribution of an inner leaflet component in a plasmamembrane alters the amount of the component in the plasma membrane,alters the relative location of the component in the inner leaflet, oralters the percentages of the component found in the inner leaflet andouter leaflet of the plasma membrane. Such a modulation may result in anincrease of the inner leaflet component concentration in the outerleaflet of the plasma membrane. Modulating tumor volume alters the tumorvolume, the volume of at least one tumor cell, or the number of tumorcells.

Methods of assaying component distribution in a plasma membrane areknown in the art and include, but are not limited to, confocalmicroscopy, atomic force microscopy, FRET, fluorescence dequenching,electron microscopy, circular dichroism, NMR, MALDI-TOF, emissionspectra analysis, light-scattering, electrospray-mass spectrometry. SeeChang et al. (1978) Anal. Biochem. 91:13-31; Kishimoto et al. (1992) J.Lipid Research 33:1255-1267; Vaccaro et al. (1995) J. Biol. Chem.270:9953-9960; and Fu et al. (1994) J. Mol. Neurosci. 5:59-67, hereinincorporated by reference in their entirety.

Methods of assaying tumor volume are known in the art and include, butare not limited to, caliper measurements, volumetry, ultrasounds,magnetic resonance imagery, ELISAs, physical examination, X-rays,positron emission tomography, bone scans, resonance Raman spectroscopy,tactile imagery, computerized tomography, and CAT scans.

As used herein, the terms “cancer”, “hyper-proliferative,” “tumor,” and“neoplastic” refer to cells having the capacity for autonomous growth,i.e., an abnormal state or condition characterized by rapidlyproliferating cell growth. Hyper-proliferative and neoplastic diseasestates may be categorized as pathologic, i.e., characterizing orconstituting a disease state, or may be categorized as non-pathologic,i.e., a deviation from normal but not associated with a disease state.The term is meant to include all types of cancerous growths or oncogenicprocesses, metastatic tissues or malignantly transformed cells, tissues,or organs, irrespective of histopathologic type or stage ofinvasiveness. The term hyper-proliferative further includes smoothmuscle cells undergoing rapid proliferating cell growth such as occursin certain cardiomyopathies. “Pathologic hyper-proliferative” cellsoccur in disease states characterized by malignant tumor growth.Examples of non-pathologic hyper-proliferative cells includeproliferation of cells associated with wound repair.

Examples of cellular proliferative and/or differentiative disordersinclude cancer, e.g., carcinoma, sarcoma, metastatic disorders orhematopoietic neoplastic disorders, e.g., leukemias. A metastatic tumorcan arise from a multitude of primary tumor types, including but notlimited to those of prostate, colon, lung, breast, and liver origin.

The terms “cancer” or “neoplasms” include malignancies of the variousorgan systems, such as those affecting the lung, breast, thyroid,lymphoid, gastrointestinal, or genitourinary tract, as well asadenocarcinomas which include malignancies such as most colon cancers,renal-cell carcinoma, prostate cancer and/or testicular tumors,non-small cell carcinoma of the lung, cancer of the small intestine andcancer of the esophagus.

The term “carcinoma” is art recognized and refers to malignancies ofepithelial or endocrine tissues including respiratory system carcinomas,gastrointestinal system carcinomas, genitourinary system carcinomas,testicular carcinomas, breast carcinomas, prostatic carcinomas,endocrine system carcinomas, and melanomas. Exemplary carcinomas includethose forming from tissue of the cervix, lung, prostate, breast, headand neck, colon and ovary. The term also includes carcinosarcomas, e.g.,which include malignant tumors composed of carcinomatous and sarcomatoustissues. An “adenocarcinoma” refers to a carcinoma derived fromglandular tissue or in which the tumor cells form recognizable glandularstructures.

The term “sarcoma” is art recognized and refers to malignant tumors ofmesenchymal derivation.

Tumors and cancers of the skin include, but are not limited to,malignant melanoma; benign epithelial tumors, including but not limitedto, seborrheic keratoses, acanthosis nigricans, fibroepithelial polyp,epithelial cyst, keratoacanthoma, and adnexal (appendage) tumors;premalignant and malignant epidermal tumors, including but not limitedto, actinic keratosis, squamous cell carcinoma, basal cell carcinoma,and merkel cell carcinoma; tumors of the dermis, including but notlimited to, benign fibrous histiocytoma, dermatofibrosarcomaprotuberans, xanthomas, and dermal vascular tumors; tumors of cellularimmigrants to the skin, including but not limited to, histiocytosis X,mycosis fungoides (cutaneous T-cell lymphoma), and mastocytosis.

Tumors and cancers of cells found in the bone marrow include, but arenot limited to, disorders arising from these cells. These disordersinclude but are not limited to the following: diseases involvinghematopoietic stem cells; committed lymphoid progenitor cells; lymphoidcells including B and T-cells; committed myeloid progenitors, includingmonocytes, granulocytes, and megakaryocytes; and committed erythroidprogenitors. These include but are not limited to the leukemias,including B-lymphoid leukemias, T-lymphoid leukemias, undifferentiatedleukemias; erythroleukemia, megakaryoblastic leukemia, monocytic;[leukemias are encompassed with and without differentiation]; chronicand acute lymphoblastic leukemia, chronic and acute lymphocyticleukemia, chronic and acute myelogenous leukemia, lymphoma, myelodysplastic syndrome, chronic and acute myeloid leukemia, myelomonocyticleukemia; chronic and acute myeloblastic leukemia, chronic and acutemyelogenous leukemia, chronic and acute promyelocytic leukemia, chronicand acute myelocytic leukemia, hematologic malignancies ofmonocyte-macrophage lineage, such as juvenile chronic myelogenousleukemia; secondary AML, antecedent hematological disorder; reactivecutaneous angioendotheliomatosis; fibrosing disorders involving alteredexpression in dendritic cells, disorders including systemic sclerosis,E-M syndrome, epidemic toxic oil syndrome, eosinophilic fasciitislocalized forms of scleroderma, keloid, and fibrosing colonopathy;angiomatoid malignant fibrous histiocytoma; carcinoma, including primaryhead and neck squamous cell carcinoma; sarcoma, including Kaposi'ssarcoma; fibroadenoma and phyllodes tumors, including mammaryfibroadenoma; stromal tumors; phyllodes tumors, including histiocytoma;T-cell lymphomas; and B-cell lymphomas.

Tumors and cancers of the heart include, but are not limited to, primarycardiac tumors, such as myxoma, lipoma, papillary fibroelastoma,rhabdomyoma, and sarcoma, and cardiac effects of noncardiac neoplasms.

Tumors and cancers of the blood vessels include, but are not limited tohemangioma, lymphangioma, glomus tumor (glomangioma), vascular ectasias,and bacillary angiomatosis, and intermediate-grade (borderline low-grademalignant) tumors, such as Kaposi sarcoma and hemangioendothelioma, andmalignant tumors, such as angiosarcoma and hemangiopericytoma.

Tumors and cancers of the B-cells include, but are not limited toprecursor B-cell neoplasms, such as lymphoblastic leukemia/lymphoma.Peripheral B-cell neoplasms include, but are not limited to, chroniclymphocytic leukemia/small lymphocytic lymphoma, follicular lymphoma,diffuse large B-cell lymphoma, Burkitt lymphoma, plasma cell neoplasms,multiple myeloma, and related entities, lymphoplasmacytic lymphoma(Waldenstrom macroglobulinemia), mantle cell lymphoma, marginal zonelymphoma (MALToma), and hairy cell leukemia.

Tumors and cancers of the liver include, but are not limited to nodularhyperplasias, adenomas, and malignant tumors, including primarycarcinoma of the liver and metastatic tumors.

Tumors and cancers of the brain include, but are not limited to gliomas,including astrocytoma, including fibrillary (diffuse) astrocytoma andglioblastoma multiforme, pilocystic astrocytoma, pleomorphicxanthoastrocytoma, and brain stem glioma, oligodendroglioma, andependymoma and related paraventricular mass lesions, neuronal tumors,poorly differentiated neoplasms, including medulloblastoma, otherparenchymal tumors, including primary brain lymphoma, germ cell tumors,and pineal parenchymal tumors, meningiomas, metastatic tumors,paraneoplastic syndromes, peripheral nerve sheath tumors, includingschwannoma, neurofibroma, and malignant peripheral nerve sheath tumor(malignant schwannoma), and neurocutaneous syndromes (phakomatoses),including neurofibromatosis, including Type 1 neurofibromatosis (NF1)and TYPE 2 neurofibromatosis (NF2), tuberous sclerosis, and VonHippel-Lindau disease.

Tumors and cancers of the ovary include, but are not limited to, ovariantumors such as, tumors of coelomic epithelium, serous tumors, mucinoustumors, endometrioid tumors, clear cell adenocarcinoma,cystadenofibroma, Brenner tumor, surface epithelial tumors; germ celltumors such as mature (benign) teratomas, monodermal teratomas, immaturemalignant teratomas, dysgerminoma, endodermal sinus tumor,choriocarcinoma; sex cord-stomal tumors such as, granulosa-theca celltumors, thecoma-fibromas, androblastomas, hill cell tumors, andgonadoblastoma; and metastatic tumors such as Krukenberg tumors.

Tumors and cancers of the kidney include, but are not limited to, benigntumors, such as renal papillary adenoma, renal fibroma or hamartoma(renomedullary interstitial cell tumor), angiomyolipoma, and oncocytoma,and malignant tumors, including renal cell carcinoma (hypernephroma,adenocarcinoma of kidney), which includes urothelial carcinomas of renalpelvis.

Tumors and cancers of the skeletal muscle include, but are not limitedto, rhabdomyosarcoma.

Tumors and cancers of the bone-forming cells include, but are notlimited to, osteoma, osteoid osteoma, osteoblastoma, osteosarcoma,osteochondroma, chondromas, chondroblastoma, chondromyxoid fibroma,chondrosarcoma, fibrous cortical defects, fibrous dysplasia,fibrosarcoma, malignant fibrous histiocytoma, Ewing sarcoma, primitiveneuroectodermal tumor, giant cell tumor, and metastatic tumors.

Tumors and cancers of the pancreas include, but are not limited to,cystic tumors and carcinoma of the pancreas; islet cell tumors,including but not limited to, insulinomas, gastrinomas, and other rareislet cell tumors.

Tumors and cancers of the breast include, but are not limited to,stromal tumors such as fibroadenoma, phyllodes tumor, and sarcomas, andepithelial tumors such as large duct papilloma; carcinoma of the breastincluding in situ (noninvasive) carcinoma that includes ductal carcinomain situ (including Paget's disease) and lobular carcinoma in situ, andinvasive (infiltrating) carcinoma including, but not limited to,invasive ductal carcinoma, no special type, invasive lobular carcinoma,medullary carcinoma, colloid (mucinous) carcinoma, tubular carcinoma,and invasive papillary carcinoma, and miscellaneous malignant neoplasms.

Tumors and cancers of the male breast include, but are not limited to,carcinoma.

Tumors and cancers of the prostate include, but are not limited to,carcinoma.

Tumors and cancers of the colon include, but are not limited to,non-neoplastic polyps, adenomas, familial syndromes, colorectalcarcinogenesis, colorectal carcinoma, and carcinoid tumors.

Tumors and cancers of the lung include, but are not limited to,bronchogenic carcinoma, including paraneoplastic syndromes,bronchioloalveolar carcinoma, neuroendocrine tumors, such as bronchialcarcinoid, miscellaneous tumors, and metastatic tumors; pleural tumors,including solitary fibrous tumors (pleural fibroma) and malignantmesothelioma.

Tumors and cancers of the thymus include, but are not limited to,thymomas, including germ cell tumors, lymphomas, Hodgkin disease, andcarcinoids. Thymomas can include benign or encapsulated thymoma, andmalignant thymoma Type I (invasive thymoma) or Type II, designatedthymic carcinoma.

Tumors and cancers of the tonsils include, but are not limited to,non-Hodgkin's lymphoma and B-cell lymphoma.

An agent of the invention comprising a prosaposin related polypeptideand an inner leaflet component (also referred to herein as “activecompounds”) can be incorporated into pharmaceutical compositionssuitable for administration to a subject. Such compositions typicallycomprise a prosaposin related polypeptide, an inner leaflet component,and a pharmaceutically acceptable carrier. In an embodiment theprosaposin related polypeptide and the inner leaflet component form ananovesicle. The nanovesicle diameter is in the range 0.01 to 10 μm,preferably 0.1 to 1 μm, more preferably 0.1 to 0.5 μm, yet morepreferably 0.2 to 0.4 μm, yet still more preferably 0.2 to 0.3 μm.Typical nanovesicle diameters include, but are not limited to, 10 nm,100 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, 200 nm, 210 nm, 220 nm,230 nm, 240 nm, 250 nm, 260 nm, 270 nm, 280 nm, 290 nm, 300 nm, 350 nm,400 nm, 450 nm, 500 nm, 550 nm, 600 nm, 650 nm, 700 nm, 750 nm, 800 nm,850 nm, 900 nm, 950 nm, and 1000 nm.

The term “administer” is used in its broadest sense and includes anymethod of introducing the compositions of the present invention into asubject. By “subject” is intended a mammal, e.g., a human, or anexperimental or animal or disease model. The subject can also be anon-human animal such as, but not limited to, a non-human primate,horse, cow, goat, pig, rabbit, mouse, guinea pig, dog, or other domesticanimal. Additionally the compositions of the invention find use in thetreatment of disorders described herein. Thus, therapies for disordersassociated with hyper-proliferating cells such as tumors or cancers areencompassed herein. “Treatment” is herein defined as the application oradministration of an agent of the invention to a patient, or applicationor administration of an agent of the invention to an isolated tissue orcell line from a patient, who has a disease or symptom of a disease,with the purpose to cure, heal, alleviate, relieve, alter, remedy,ameliorate, improve, or affect the disease or symptoms of the disease.

As used herein the language “pharmaceutically acceptable carrier” isintended to include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration. Theuse of such media and agents for pharmaceutically active substances iswell known in the art. Except insofar as any conventional media or agentis incompatible with the active compound, use thereof in thecompositions is contemplated. Supplementary active compounds can also beincorporated into the compositions.

An anti-tumor agent or pharmaceutical composition of the invention isformulated to be compatible with its intended route of administration.Examples of routes of administration include parenteral, e.g.,intravenous, intradermal, subcutaneous, oral (e.g., inhalation),transdermal (topical), transmucosal, intraperitoneal, and rectaladministration. Solutions or suspensions used for parenteral,intradermal, or subcutaneous application can include the followingcomponents: a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerin, propylene glycolor other synthetic solvents; antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes, or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF; Parsippany, N.J.), or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion, and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride, inthe composition. Prolonged absorption of the injectable compositions canbe brought about by including in the composition an agent that delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompounds (e.g., a prosaposin-related polypeptide and an inner leafletcomponent) in the required amount in an appropriate solvent with one ora combination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying, which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth, or gelatin; an excipientsuch as starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring. For administrationby inhalation, the compounds are delivered in the form of an aerosolspray from a pressurized container or dispenser that contains a suitablepropellant, e.g., a gas such as carbon dioxide, or a nebulizer.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated with each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. Depending on thetype and severity of the disease, about 1 μg/kg to about 15 mg/kg (e.g.,0.1 to 20 mg/kg) of an agent of the invention is an initial candidatedosage for administration to the patient, whether, for example, by oneor more separate administrations, or by continuous infusion. A typicaldaily dosage might range from about 1 μg/kg to about 100 mg/kg or more,depending on the factors mentioned above. For repeated administrationsover several days or longer, depending on the condition, the treatmentis sustained until a desired suppression of disease symptoms occurs.However, other dosage regimens may be useful. The progress of thistherapy is easily monitored by conventional techniques and assays. Anexemplary dosing regimen is disclosed in WO 94/04188. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art. Thecompounds can also be prepared in the form of suppositories (e.g., withconventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

The anti-tumor or anti-cancer agents described herein can beadministered transdermally. Transdermal administration typicallyinvolves the delivery of a pharmaceutical agent for percutaneous passageof the drug into the systemic circulation of the subject or patient. Theskin sites include anatomic regions for transdermally administering thedrug and include the forearm, abdomen, chest, back, buttock, mastoidalarea, and the like.

Transdermal delivery is accomplished by exposing a source of the agentor complex to a patient's skin for an extended period of time.Transdermal patches have the added advantage of providing controlleddelivery of a pharmaceutical agent to the body (see Hadgraft and Guy(eds) (1989) Transdermal Drug Delivery: Developmental Issues andResearch Initiatives, Marcel Dekker, Inc.; Robinson & Lee (eds) (1987)Controlled Drug Delivery: Fundamentals and Applications, Marcel Dekker,Inc; and Kydonieus & Berner (eds) (1987) Transdermal Delivery of Drugsvols 1-3, CRC Press, herein incorporated by reference). Such dosageforms can be made by dissolving, dispersing, or otherwise incorporatingthe combination of saposin C related polypeptide anddioleoylphosphatidylserine (DOPS) in a proper medium, such as anelastomeric matrix material. Absorption enhancers can also be used toincrease the flux of the compound across the skin. The rate of such fluxcan be controlled by providing a rate-controlling membrane or dispersingthe agent in a polymer matrix or gel.

A variety of types of transdermal patches will find use in the methodsdescribed herein. For example, a simple adhesive patch can be preparedfrom a backing material and an acrylate adhesive. The pharmaceuticalagent and any enhancer are formulated into the adhesive casting solutionand allowed to mix thoroughly. The solution is cast directly onto thebacking material and the casting solvent is evaporated in an oven,leaving an adhesive film. The release liner can be attached to completethe system.

Alternatively a polyurethane matrix patch can be employed to deliver theagent. The layers of this patch comprise a backing, a polyurethanedrug/enhancer matrix, a membrane, an adhesive, and a release liner. Thepolyurethane matrix is prepared using a room temperature curingpolyurethane prepolymer. Addition of water, alcohol, and complex to theprepolymer results in the formation of a tacky firm elastomer that canbe directly cast on the backing material.

A further embodiment of this invention will utilize a hydrogel matrixpatch. Typically, the hydrogel matrix will comprise alcohol, water,drug, and several hydrophilic polymers. This hydrogel matrix can beincorporated into a transdermal patch between the backing and theadhesive layer.

For passive delivery systems, the rate of release is typicallycontrolled by a membrane placed between the reservoir and the skin, bydiffusion from a monolithic device, or by the skin itself serving as arate-controlling barrier in the delivery system (see U.S. Pat. Nos.4,816,258; 4,927,408; 4,904,475; 4,588,580; 4,788,062, hereinincorporated by reference). The rate of drug delivery will be dependentin part upon the nature of the membrane. For example, the rate of drugdelivery across membranes within the body is generally higher thanacross dermal barriers. The rate at which the agent is delivered fromthe device to the membrane is most advantageously controlled by the useof rate-limiting membranes placed between the reservoir and the skin.When the skin is sufficiently permeable to the complex (i.e., absorptionthrough the skin is greater than the rate of passage through themembrane), the membrane will serve to control the dosage rateexperienced by the patient.

Suitable permeable membrane materials may be selected based on thedesired degree of permeability, the nature of the agent, and themechanical considerations related to constructing the device. Exemplarypermeable membrane materials include a wide variety of natural andsynthetic polymers, such as polydimethylsiloxanes (silicone rubbers),ethylenevinylacetate copolymer (EVA), polyurethanes,polyurethane-polyether copolymers, polyethylenes, polyamides,polyvinylchlorides (PVC), polypropylenes, polycarbonates,polytetrafluoroethylenes (PTFE), cellulosic materials, e.g., cellulosetriacetate and cellulose nitrate/acetate, and hydrogels, e.g.,2-hydroxyethylmethacrylate (HEMA).

Other items may be contained in the device, such as otherpharmaceutically acceptable carriers, depending on the desired devicecharacteristics. For example, the compositions according to thisinvention may also include one or more preservatives or bacteriostaticagents, e.g., methyl hydroxybenzoate, propyl hydroxybenzoate,chlorocresol, benzalkonium chlorides, and the like. These pharmaceuticalcompositions also can contain other active ingredients such asantimicrobial agents, particularly antibiotics, anesthetics, andantipruritic agents.

Another aspect of this invention provides for the topical delivery of anagent of the invention. This treatment regimen is suitable either forthe systemic administration of the anti-tumor agent or for localizedtherapy, i.e., directly to pathological or diseased tissue.

Typically, the topical formulations will comprise a preparation fordelivering the agent directly to the affected area comprising thecomplex, typically in concentrations in the range of from about 0.001%to 10%; preferably, from about 0.01 to about 10%; more preferably fromabout 0.1 to about 5%; and most preferably from about 1 to about 5%,together with a non-toxic, pharmaceutically acceptable topical carrier(Barry (eds). Dermatological Formulations: Percutaneous Absorption(1983) Marcel Dekker, Inc; for standard dosages of conventionalpharmaceutical agents see, e.g., Physicians Desk Reference (1992Edition); and American Medical Association (1992) Drug EvaluationsSubscriptions).

Topical preparations can be prepared by combining the agent withconventional pharmaceutical diluents and carriers commonly used intopical dry, liquid, cream, and aerosol formulations. Ointment andcreams may, for example, be formulated with an aqueous or oily base withthe addition of suitable thickening and/or gelling substances. Suchbases may include water and/or an oil such as liquid paraffin or avegetable oil such as peanut oil or castor oil. Thickening agents whichmay be used according to the nature of the base include soft paraffin,aluminum stearate, cetostearyl alcohol, propylene glycol, polyethyleneglycols, wool fat, hydrogenated lanolin, beeswax, and the like. Lotionsmay be formulated with an aqueous or oily base and will, in general,also include one or more of the following: stabilizing agents,emulsifying agents, dispersing agents, suspending agents, thickeningagents, coloring agents, perfumes, and the like. Powders may be formedwith the aid of any suitable base, e.g., talc, lactose, starch, and thelike. Drops may be formulated with an aqueous base or non-aqueous basealso comprising one or more dispersing agents, suspending agents,solubilizing agents, and the like.

Dosage forms for the topical administration of an agent of thisinvention include powders, sprays, ointments, pastes, creams, lotions,gels, solutions, patches, and inhalants. The active compound may bemixed under sterile conditions with a pharmaceutically acceptablecarrier, and with any preservatives, buffers, or propellants which maybe required.

The ointments, pastes, creams, and gels also may contain excipients,such as animal and vegetable fats, oils, waxes, paraffins, starch,tragacanth, cellulose derivatives, polyethylene glycols, silicones,bentonites, talc, and zinc oxide, or mixtures thereof. Powders andsprays also can contain excipients such as lactose, talc, aluminumhydroxide, calcium silicates, and polyamide powder, or mixtures of thosesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons suchas butane and propane.

The methods of the present invention are also applicable to the deliveryof pharmaceutical agents through mucosal membranes such as agastrointestinal, sublingual, buccal, nasal, pulmonary, vaginal,corneal, and ocular membranes (Mackay et al. (1991) Adv. Drug Del. Rev.7:313-338).

For delivery to the buccal or sublingual membranes, typically an oralformulation such as a lozenge, tablet, or capsule will be used. Themethod of manufacture of these formulations are known in the art,including, but not limited to, the addition of the agent to apre-manufactured tablet; cold compression of an inert filler, a binder,and encapsulation.

Another oral formulation is one that can be applied with an adhesivesuch as the cellulose derivative, hydroxypropyl cellulose, to the oralmucosa, for example as described in U.S. Pat. No. 4,940,587,incorporated by reference. This buccal adhesive formulation, whenapplied to the buccal mucosa, allows for the controlled release of anagent into the mouth and through the buccal mucosa.

For delivery to the nasal and/or pulmonary membranes, typically anaerosol formulation will be employed. The term “aerosol” includes anygas-borne suspended phase of an agent of the invention which is capableof being inhaled into the bronchioles or nasal passages. Specifically,aerosol includes a gas-borne suspension of droplets of the compounds ofthe instant invention, as may be produced in a metered dose inhaler ornebulizer, or in a mist sprayer. Aerosol also includes a dry powdercomposition of the agent suspended in air or other carrier gas, whichmay be delivered by inhalation from an inhaler device.

The compositions of the invention are useful to treat any of thedisorders discussed herein. The compositions are provided intherapeutically effective amounts. By “therapeutically effectiveamounts” is intended an amount sufficient to modulate the desiredresponse. As defined herein, a therapeutically effective amount ofprotein or polypeptide in the agent (i.e., an effective dosage) rangesfrom about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight,and even more preferably about 1 to 15 mg/kg. A therapeuticallyeffective amount of an inner leaflet component in the agent (i.e., aneffective dosage) ranges from about 0.001 to 30 mg/kg body weight,preferably from about 0.01 to about 30 mg/kg body weight, morepreferably about 0.01 to about 20 mg/kg body weight, yet more preferably0.01 to 10 mg/kg body weight, and even more preferably about 0.1 to 9mg/kg, 0.1 to 8 mg/kg, 0.1 to 7 mg/kg, 0.1 to 6 mg/kg, 0.1 to 5 mg/kg,0.1 to 4 mg/kg, or 0.1 to 3 mg/kg body weight.

The molar ratio of the polypeptide to the inner leaflet component in anagent of the invention is in the range from about 1:1 to about 1:50,preferably about 1:1 to about 1:25, more preferably about 1:1 to about1:10, yet more preferably about 1:7 or about 1:3. Suitable ratiosinclude, but are not limited to, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8,1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20,1:21, 1:22, 1:23, 1:24, 1:25, 1:30, 1:35, 1:40, 1:45, and 1:50. The massratio of the polypeptide to the inner leaflet component in an agent ofthe invention is in the range from about 15:1 to about 3:10, preferablyabout 15:1 to about 3:5, more preferably about 15:2 to about 3:0, yetmore preferably about 15:7 or about 5:1. It is recognized that thepreferred ratio of the polypeptide and inner leaflet component in anagent of the invention may be affected by certain factors such as, butnot limited to, the target cell type.

The skilled artisan will appreciate that certain factors may influencethe dosage required to effectively treat a subject, including but notlimited to the severity of the disease or disorder, previous treatments,the general health and/or age of the subject, and other diseasespresent. Moreover, treatment of a subject with a therapeuticallyeffective amount of a protein, polypeptide, or antibody can include asingle treatment or, preferably, can include a series of treatments. Ina preferred example, a subject is treated with a therapeuticallyeffective amount of the agent one time per week for between about 1 to10 weeks, preferably between 2 to 8 weeks, more preferably between about3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. Itwill also be appreciated that the effective dosage of antibody, protein,or polypeptide used for treatment may increase or decrease over thecourse of a particular treatment. Changes in dosage may result andbecome apparent from the results of diagnostic assays as describedherein.

Where a subject undergoing therapy exhibits a partial response or arelapse following a prolonged period of remission, subsequent course oftreatment with an agent of the invention may be administered. Thus,subsequent to a period of time off from a first treatment period, whichmay have comprised a single dosing regimen or a multiple dosing regimen,a subject may receive one or more additional treatment periodscomprising single or multiple dosing regimens. Such a period of time offbetween treatment periods is referred to herein as a time period ofdiscontinuance. It recognized that the length of the time period ofdiscontinuance is dependent upon the degree of tumor response achievedwith any prior treatment periods with the anti-tumor agents of theinvention.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

The results of treatment of a cancer may be assayed by any method knownto one skilled in the art including, but not limited to, physicalexamination, laboratory, nuclear, and radiographic studies (i.e.computer tomography and/or magnetic resonance imagery), ultrasound andother procedures.

As used herein “cell death” refers to loss of cell life through anymechanism including apoptosis, necrosis, and lysis. By “apoptosis” or“programmed cell death” is intended a normal physiological processrequiring regulated metabolic activity by the dying cell, oftencharacterized by cell shrinkage, chromatin condensation, and/or nuclearfragmentation, loss of membrane integrity, DNA fragmentation, and/orcompromised or blebbing of plasma membranes.

The following examples are offered by way of illustration and notlimitation.

EXPERIMENTAL Example 1 Purification of Recombinant Saposin C

Recombinant saposin C was overexpressed in E. coli cells by using theisopropyl-1-thio-β-D-galactopyranoside inducing pET system (Qi et al.(1994) J. Biol. Chem. 269:16746-16753, herein incorporated by referencein its entirety). Expressed polypeptides with a His-tag were eluted fromnickel columns. After dialysis, the polypeptides were further purifiedby HPLC chromatography as follows. A C4 reverse phase column wasequilibrated with 0.1% trifluoroacetic acid (TFA) for 10 minutes. Theproteins were eluted in a linear (0-100%) gradient of 0.1% TFA inacetonitrile over 60 minutes. The major protein peak was collected andlyophilized. Protein concentration was determined as previouslydescribed (Qi et al. (1994) J. Biol. Chem. 269:16746-16753).

Example 2 Bath Sonication of Saposin C and Dioleoylphosphatidylserine

Dioleoylphosphatidylserine (DOPS) was obtained from Avanti Polar Lipids(Alabaster Ala.). Twenty to thirty μmoles of DOPS in chloroform weredried under N₂ and vacuum to lipid films. Five to ten μmoles saposin Cpolypeptide was added to the dried films and suspended in 50 μlMcIlvanine buffer (pH 4.7). The suspension was then brought to a 1 mlvolume with either cell culture medium or phosphate buffered saline(PBS) (Ausubel et al. (2002) Current Protocols in Molecular Biology.John Wiley & Sons, New York, N.Y., herein incorporated by reference).The mixture was sonicated in a bath sonicator for approximately 20minutes. Ice was added as needed to prevent overheating the samples.

Example 3 Tissue Culture Conditions

Squamous cell carcinoma (SCC) cells and L5178Y cells were cultured inDEME medium (Gibco) supplemented with 10% FBA. Normal ImmortalizedKeratinocytes (NIK) cells were grown in 50% Cascade medium 154 (CascadeBiologics) and 50% Keratinocyte-SFM medium (Gibco).

Example 4 Ex Vivo Analysis of Effects of Saposin C-DOPS on SCC Cells

Squamous cell carcinoma (SCC) cells and control (NIK cells) were grownin media described elsewhere herein. NIKs were used as a control, sinceSCC have been suggested to develop through a multistep process in humanskin keratinocytes (Kubo et al. (2002) J. Med. Invest. 49:111-117).Culture medium was removed from established plates of NIK and SCC cells.Culture medium containing no treatment, saposin C, DOPS, or 8 μM saposinC+26 μM DOPS was added to established plates of NIK and SCC cells. Thecells were examined 48-72 hours after treatment. Results from one suchexperiment are presented in FIG. 1.

Squamous cell carcinoma (SCC) cells were grown in media describedelsewhere herein. Culture medium was removed from established plates ofSCC cells. Culture medium containing no treatment or an agent comprising10 μM saposin C+30 μM DOPS was added to established plates of SCC cells.The cells were incubated for 24 hours and analyzed by TUNEL staining,gel electrophoresis of genomic DNA, or hybridization assays with theanti-coagulant protein, annexin V, data not shown.

Example 5 Ex Vivo Analysis of Saposin C-DOPS Effect on Murine LymphomaCells

Tissue culture plates were seeded with mouse L5178Y-R lymphoma cells.After establishment of the cultures, the culture media was removed andthe cells were washed. The cells were overlaid with DEME+10% FBAsupplemented with either no drug, 60 μM DOPS, 20 μM saposin C, or 10 μMsaposin C and 30 μM DOPS. The cultures were incubated for 24-48 hours.Cultures were examined after the incubation period. Results from onesuch experiment are presented in FIG. 2.

Example 6 In Vivo Analysis of Saposin C-DOPS Effect on Tumor Volume

Nude mice were maintained in accord with the Cincinnati Children'sResearch Foundation guidelines governing the care of laboratory mice.Two groups of five nude mice were injected on the up-back with 2×10⁶SCCs subcutaneously to initiate tumor growth. Two tumors wereestablished in each mouse. The tumors were allowed to establish for 21days. On day 21 the animals received a subcutaneous injection at thetumor site of either the PBS diluent alone or an agent comprisingsaposin C (10 mg/kg body weight) and DOPS (2 mg/kg body weight). On day27 the animals received a second subcutaneous injection at the tumorsite of either the PBS diluent alone or an agent comprising saposin C(10 mg/kg body weight) and DOPS (2 mg/kg body weight). Tumor sizes weremeasured every other day with a caliper and volumes were estimatedaccording to the formula V=(π/4)LW². Results obtained from one suchexperiment are presented in FIG. 3, panel A.

In a separate set of experiments, nude mice were maintained in accordwith the Cincinnati Children's Research Foundation guidelines governingthe care of laboratory mice. Two groups of five nude mice were injectedon the up-back with 2×10⁶ SCCs subcutaneously to initiate tumor growth.Two tumors were established in each mouse. The tumors were allowed toestablish for 6 days. On day 6 the animals received a subcutaneousinjection at the tumor site of either the PBS diluent alone or an agentcomprising saposin C (10 mg/kg body weight) and DOPS (2 mg/kg bodyweight). Tumor sizes were measured every other day with a caliper andvolumes were estimated according to the formula V=(π/4)LW². Resultsobtained from one such experiment are presented in FIG. 3, panel B.

Example 7 Effect of Saposin C-DOPS on Human Squamous Cell CarcinomaTumor Tissue

Mouse xenografts were prepared with SCC tumors by methods known in theart. The fluorescent label nitrobenzoxadiazole (NBD) was linked tophosphatidylserine and a mixture of NBD-PS and DOPS was prepared. TheNBD-DOPS was used to prepare a fluorescently labeled NBD-PS/DOPS/SaposinC complex. NBD-PS/DOPS was injected into the tumor at 0.1 mg NBD-PS/kgbody weight and 2 mg DOPS/kg body weight. NBD-PS/DOPS/Saposin C wasinjected into the tumor at 0.1 mg NBD-PS/kg body weight, 2 mg DOPS/kgbody weight, and 10 mg saposin C/kg body weight. Tumors were harvested24 hours after administration of the agent. Microsections of the tumorswere examined for fluorescence. Results from such an experiment areshown in FIG. 4.

Example 8 Ex Vivo Analysis of Saposin C-DOPS Effect on Human Cells

Cells from human cancer tissue and healthy tissue were grown in mediasuitable to the cell line. Cells from the following cancer tissue andhealthy tissue cell lines were analyzed: Breast cancer: MCF-7, MCF-7transfected with a dominant negative caspase 9, MCF-7 transfected with avector control, BT-549; Head and neck: SCC-25, FaDu; Melanomas: MeWo,Sk-Mel-28; Leukemias: K-562, HL60; Cervical cancer: Hela; Ovariancancer: PA1, PA1 transfected with a dominant negative caspase 9, PA1-E6;SK-OV3; Prostate cancer: DU145, PC3; Neuroblastomas: SK-N-SH, SK-SY-5Y,CHLA-79; Ewing sarcoma: 5838; T cell lymphomas; Rodu T; GCT; Lungcancer: A549,H441; Liver cancer: HepG2; Healthy breast: MCF-10A; andHealthy keratinocytes: NIK. 96-well flat-bottom tissue culture plates(Falcon, Becton-Dickson Labware, Franklin Lakes N.J.) were seeded withcells at a density of 10⁴ cells per well. Cells were plated in 100 tlcomplete medium with or without an agent of the invention.

The conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (available from Sigma) to a formazan product (18992) was used toassess viable cell density. Seventy-two hours after culturing the cells,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) wasadded to each well to a final concentration of 0.25 mg/ml. The plateswere incubated at 37° C. for 3 hours in the dark. The reactions wereterminated by the addition of 0.04 N HCl in isopropanol. The plates werethoroughly mixed and analyzed at 570 nm on a micro ELISA plate reader(SpectraMax Plus, Molecular Devices, Sunnyvale Calif.). Growth curvesand regression analysis were performed using PharmTools Pro computersoftware (The McCary Group, Elkins Park, Pa.).

Human Cells Cell Death (%) Cancer Cell Lines Non-treated Treated KnownGene Defects Breast: MCF-7  6.8 ± 3.5 96.1 ± 1.0 Caspase-3 null MCF-7(transfected)  7.5 ± 3.5 18.9 ± 6.8 Caspases-9 DN MCF-7 (transfected)10.7 ± 3.7 94.0 ± 0.7 Vector control BT-549  6.9 ± 2.8 33.6 ± 6.6 p53mutation Head & neck: SCC-25  7.4 ± 2.8 57.8 ± 4.6 p53 null FaDu 14.6 ±5.7 91.0 ± 0.2 Melanomas: MeWo  8.3 ± 2.6 81.9 ± 5.4 p53 mutationSK-Me1-28  7.7 ± 3.2 76.2 ± 2.0 Apaf-1 & p53 mutations Leukemia: K-562 7.0 ± 2.8 58.0 ± 3.4 Apaf-1 & p53 nulls HL-60 21.2 ± 5.4 44.2 ± 3.6 p53null Cervix: Hela  3.8 ± 1.1 47.3 ± 4.4 p53 mutation Ovarian: PA1  7.1 ±1.9 89.9 ± 0.2 PA1 (transfected)  8.3 ± 3.0 54.1 ± 2.5 Caspase-9 DNPA1-E6 10.6 ± 3.9 71.2 ± 2.7 p53 mutation SK-OV3 11.8 ± 5.9 47.4 ± 12.1Prostate DU145  4.0 ± 1.2 37.0 ± 2.8 PC3  9.0 ± 2.6 48.9 ± 16.8Neuroblastomas: 12.4 ± 4.6 51.7 ± 19.1 Caspase-8 mutation SK-N-SHSK-SY-5Y  3.7 ± 0.6 68.0 ± 12.3 Caspase-8 mutation CHLA-79  3.6 ± 0.652.5 ± 6.5 Ewing sarcoma: 5838 12.8 ± 3.7 72.1 ± 3.9 T cell lymphomas12.1 ± 2.0 77.2 ± 5.8 Rodu T  6.1 ± 2.9 22.2 ± 8.5 GCT  3.4 ± 1.4 26.6 ±1.4 Lung A549  5.0 ± 0.8 25.2 ± 13.7 p53 mutation H441 10.1 ± 4.0 25.4 ±2.2 p53 mutation Liver: HepG2  9.4 ± 3.1 22.0 ± 10.2 Normal CellsBreast: MCF-10A 12.8 ± 5.2 19.5 ± 5.9 Keratinocyte: NIK 16.1 ± 8.4 18.2± 6.7

Example 9 Evaluation of the Saposin C/DOPS IC₅₀

Mixtures of Saposin C and DOPS at molar ratios of 1:7, 1:3, and 1:10were prepared. Polypeptides comprised of various fragments of theSaposin C protein were prepared as described previously (Wang et al.(2003) Arch. Biochem. & Biophys. 415:45-53, herein incorporated byreference in its entirety). The mutant Saposin C polypeptides are asfollows: HNSC is comprised of amino acid residues 1-40; H1 is comprisedof residues 4-20; and H-2 is comprised of amino acid residues 24-40.

Human SK-Mel-28 Melanoma cells were cultured on 96 well flat bottomedtissue culture plates. The cells were covered with media containingvarious concentrations of saposin C, DOPS, HNSC:DOPS (1:3), H-1:DOPS1:3; H-2:DOPS 1:1; or a mixture of full length Saposin C:DOPS at 1:7,1:3, or 1:10. Each treatment was administered to quadruplicate plates ofSK-Mel-28 cells. Cell inhibition was analyzed using the MTT conversionassayed described elsewhere herein. The data were analyzed byfundamental linear regression using PharmTools Pro computer software(The McCary Group, Elkins Park, Pa.). Results are presented in Table 2.

TABLE 2 SK-Mel-28 Melanomas IC₅₀ Samples Saposin C DOPS Saposin C: DOPS(1:7) 19.5 ± 11.0 136.6 ± 78.0  Saposin C: DOPS (1:3) 99.8 ± 13.0 299.3± 39.0  Saposin C: DOPS (1:10) 81.3 ± 13.2  8134 ± 132.3 Saposin C only786.0 ± 25.4  DOPS only 14193.4 ± 1886.0  HNSC (saposin C(1-40)): 211 ±32   633 ± 96.0 DOPS (1:3) H-1 (saposin C helix-1): 327 ± 36    981 ±108.0 DOPS (1:3) 11-2 (saposin C helix-2): 243 ± 20   729 ± 60.0 DOPS(1:3)

Example 10 In Vivo Analysis of Saposin C-DOPS Effect on Tumor Cells

Nude mice were maintained in accord with the Cincinnati Children'sResearch Foundation guidelines governing the care of laboratory mice.Mice were injected on the up-back with 2×10⁶ SCCs subcutaneously toinitiate tumor growth. The tumors were allowed to establish. The animalswere treated with either DOPS (2 mg/kg body weight) or an agentcomprising saposin C (10 mg/kg body weight) and DOPS (2 mg/kg bodyweight). Forty-eight hours after administration of the treatment, thetumors were harvested.

Tissue sections were prepared from the tumors and examined using avariety of methods.

Tissue sections were examined by terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)to evaluate apoptosis. (Results of one such experiment are shown in FIG.5, Panels A and B.)

Tissue sections were stained with hematoxylin and eosin. (Results of onesuch experiment are shown in FIG. 5, Panels C and D.)

All publications, patents, and patent applications mentioned in thespecification are indicative of the level of those skilled in the art towhich this invention pertains. All publications, patents, and patentapplications are herein incorporated by reference to the same extent asif each individual publication or patent application was specificallyand individually incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

That which is claimed: 1-54. (canceled)
 55. A method for modulatingtumor volume in a subject comprising the step of administering to thesubject a composition comprising a phospholipid, wherein thephospholipid is selected from the group consisting ofphosphatidylserine, phosphatidylethanolamine, and structural analogsthereof; an isolated saposin C-related polypeptide, wherein thepolypeptide has an amino acid sequence at least 60 percent identical toSEQ ID NO: 2; and wherein the phospholipid forms a nanovesicleincorporating the polypeptide.
 56. The method of claim 55, wherein thepolypeptide has an amino acid sequence at least 80 percent identical toSEQ ID NO:
 2. 57. The method of claim 55, wherein the compositionfurther comprises a pharmaceutically acceptable carrier.
 58. The methodof claim 55, wherein the composition further comprises an additionalactive ingredient.
 59. The method of claim 55, wherein the phospholipidis selected from the group consisting of phosphatidylserine,phosphatidylethanolamine, phosphatidic acid, phosphatidylglycerol,phosphatidylinositol, palmitoyloleoylphosphatidylserine,palmitelaidoyloleoylphosphatidylserine,myristoleoyloleoylphosphatidylserine, dilinoleoylphosphatidylserine,palmiticlinoleoylphosphatidylserine, lysophosphatidylserine, anddioleoylphosphatidylserine.
 60. The method of claim 55, wherein theadministration is selected from the group consisting of parenteral,intradermal, subcutaneous, oral, transdermal, transmucosal,intraperitoneal, and rectal.
 61. The method of claim 55, wherein theadministration is given as a single dose.
 62. The method of claim 55,wherein the administration is given as a multiple dose.
 63. The methodof claim 55, wherein the modulation results in a decrease in tumorvolume.
 64. The method of claim 55, wherein the tumor is a brain tumor.65. The method of claim 55, wherein the tumor is a pancreatic tumor. 66.A method for treating a cancer in a subject comprising the step ofadministering to the subject a composition comprising a phospholipid,wherein the phospholipid is selected from the group consisting ofphosphatidylserine, phosphatidylethanolamine, and structural analogsthereof; an isolated saposin C-related polypeptide, wherein thepolypeptide has an amino acid sequence at least 60 percent identical toSEQ ID NO: 2; and wherein the phospholipid forms a nanovesicleincorporating the polypeptide.
 67. The method of claim 66, wherein thepolypeptide has an amino acid sequence at least 80 percent identical toSEQ ID NO:
 2. 68. The method of claim 66, wherein the compositionfurther comprises a pharmaceutically acceptable carrier.
 69. The methodof claim 66, wherein the composition further comprises an additionalactive ingredient.
 70. The method of claim 66, wherein the phospholipidis selected from the group consisting of phosphatidylserine,phosphatidylethanolamine, phosphatidic acid, phosphatidylglycerol,phosphatidylinositol, palmitoyloleoylphosphatidylserine,palmitelaidoyloleoylphosphatidylserine,myristoleoyloleoylphosphatidylserine, dilinoleoylphosphatidylserine,palmiticlinoleoylphosphatidylserine, lysophosphatidylserine, anddioleoylphosphatidylserine.
 71. The method of claim 66, wherein theadministration is selected from the group consisting of parenteral,intradermal, subcutaneous, oral, transdermal, transmucosal,intraperitoneal, and rectal.
 72. The method of claim 66, wherein thecancer is a brain cancer.
 73. The method of claim 66, wherein the canceris a pancreatic cancer.
 74. A process for the manufacture of apharmaceutical agent comprising the steps of: (a) preparing acomposition in a pharmaceutically acceptable carrier wherein thecomposition comprises (i) a phospholipid, wherein the phospholipid isselected from the group consisting of phosphatidylserine,phosphatidylethanolamine, and structural analogs thereof and (ii) anisolated saposin C-related polypeptide, wherein the polypeptide has anamino acid sequence at least 60 percent identical to SEQ ID NO: 2; and(b) treating the composition to form a nanovesicle.